ABSTRACT- Introduction: The impact of tumor hypoxia on local tumor control has long been a concern for solid tumors treated with radiation therapy. It has been proposed that tumor microenvironment, with fluctuating hypoxia, low pH and nutrient deprivation may be responsible for diminished DNA repair and increased mutagenesis. We hypothesized that hypoxia alone could lead to genomic alterations. Methods/Results: To investigate our hypothesis we used the AL mutation assay. AL cells are human-hamster hybrid cells, containing standard set of CHO-K1 chromosomes and single copy of human chromosome 11. Chromosome 11 encodes cell surface antigenic markers CD59 that render AL cells sensitive to killing by a specific monoclonal antibody in the presence of complement. Since the human chromosome is not essential for viability of the hybrid cell, except for a small segment on the tip of short arm, mutations ranging from point mutation to loss of almost whole chromosome 11 can be detected. After exposing AL cells to different concentrations and durations of oxygen, we obtained survival curves for each condition. The significant number of mutated cells was discovered in extreme hypoxic conditions, ranging from 0-0.1%O2. To qualitate mutations from these experiments, polymerase chain reaction analysis was performed on RAS at 11p15.5, LDHA at 11p15.4, CAT at 11p13, Centromere and API at 11q23-24. No delitions were detected on human chromosome 11. All four exons of CD59 gene were expressed. Conclusions: This study provided confirmation that hypoxia itself causes mutations in cells exposed to conditions found in tumor microenvironment. Significant mutagenesis is induced in extreme hypoxia, at a rate of about 130 mutants per 105 surviving cells. PCR analyses indicate that CD59- mutants could result from point mutations to very small deletions. This study was supported in part by CA42745 and CA0936.